Derivatives of kanamycin

ABSTRACT

Derivatives of kanamycin A and B have been prepared which possess substantially improved anti-bacterial activity. An example of such an agent is 1-(L-(-)- delta -amino- Alpha hydroxyvaleryl)-kanamycin A (IVa, BB-K23).

United States Patent 1191 Naito et al.

1451 May27, 1975 1 DERIVATIVES OF KANAMYCIN [75] Inventors: TakayukiNaito; Susumu Nakagawa;

Yoshio Abe, all of Tokyo, Japan [73] Assignee: Bristol-Myers Company,New York,

[22] Filed: Feb. 23, 1973 [21] Appl. No.: 335,210

[52] U.S. Cl... 260/210 K; 260/210 AB; 260/210 R;

424/180 [51] Int. Cl. C07c 47/18 [58] Field of Search 260/210 AB, 210 K,210 R [56] References Cited UNITED STATES PATENTS 3,032,547 5/1962Rothrock et al. 260/210 K 3,541,078 11/1970 Woo et al. 260/210 R3,753,973 8/1973 Umazawa et a] 260/210 K 3,781,268 12/1973 Kawaguchi etal. 260/210 AB Primary Examiner.1ohnnie R. Brown Attorney, Agent, orFirmRobert E. Havranek [S 7 ABSTRACT 7 Claims, No Drawings DERIVATIVESOF KANAMYCIN BACKGROUND OF THE INVENTION 1. Field of the Invention Thisinvention relates to a semisynthetic lsubstituted derivatives ofkanamycin A or B, said compounds being prepared by acylating thel-aminofunction of kanamycin A or B with a S-amino-ahydroxyvalerylmoiety.

2. Description of the Prior Art The kanamycins are known antibioticsdescribed in Merck Index, 8th Edition, pp. 597598. Kanamycin A is acompound having the formula CH -NH Kanamycin B is a compound having theformula HO CH -NH The compound designatedl-[L-(-)-Y-amino-ahydroxybutyryllkanamycin A [BB-K8] is described in theJournal of Antibiotics, 25(12), pp 695-731 (December 1972).

SUMMARY OF THE INVENTION The compound having the formula (Ia) or Kanamycin B Kanamyc in A in which R is OH or NH and R isL-()-8-amino-ahydroxyvaleryl or a nontoxic pharmaceutically acceptableacid addition salt thereof is a valuable antibacterial agent.

This invention relates to semi-synthetic derivatives of kanamycin A andB, said compounds being known as l-[L-()-S-amino-oz-hydroxyvaleryl]kanamycin A and B having the formula in which R is OH or NH and R is L-()-8-amino-ahydroxyvaleryl; or a nontoxic pharmaceutically acceptableacid addition salt thereof.

For the purpose of this disclosure, the term nontoxic, pharmaceuticallyacceptable acid addition salt shall mean a mono, di-, tri-, tetra, orpentasalt formed by the interaction of 1 molecule of compound IV with 71-5 moles of a nontoxic, pharmaceutically acceptable acid. Includedamong these acids are acetic, hydrochloric, sulfuric, maleic,phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, andthose other acids commonly used to make salts of amine containingpharmaceuticals.

The compounds of the present invention are prepared by the followingdiagramatic scheme:

N- (Benzyloxycarbonyloxy) Succinimide [6 -Monobenz .lox

922w AorB (II) 2.) Compound II N-Hydroxysuccinimide ester ofL-(-)-6-benzyloxycarbonylr amino-a-hydroxyv-aleric acid E c=o I in! IIICH I 2 0 E :1 cn -mx-c-o-ca -c n 5.) Compound III H /Pd/C HO CH -NH Apreferred embodiment of the present invention is the compound having theformula in which R is H or R is H or L-()-8-amino-a-hydroxyvaleryl and Ris OH or NH wherein R or R must be other than H; or a nontoxicpharmaceutically acceptable acid addition salt thereof.

Another preferred embodiment is the compound of formula V in which R isH C H CH O-C- R is H and R is OH or NH A more preferred embodiment isthe compound of formula V in which R is L-()-6-amino-ahydroxyvaleryl, Ris hydrogen and R is OH or NH A most preferred embodiment is thecompound of formula V wherein R is H, R is L-(-)-8-amino-ahydroxyvaleryland R is OH; or a nontoxic pharmaceutically acceptable acid additionsalt thereof.

Another most preferred embodiment is the compound of formula V wherein Ris H, R is L-()-8- amino-a-hydroxyvaleryl and R is NH or a nontoxicpharmaceutically acceptable and addition salt thereof.

Other most preferred embodiments are the sulfate, hydrochloride,acetate, maleate, citrate, ascorbate, nitrate or phosphate salts ofcompound V.

Another more preferred embodiment is the monosulfate salt of compound V.

Still another preferred embodiment is the disulfate salt of compound V.

The objectives of the present invention have been achieved, by theprovision according to the present invention of the process for thepreparation of the compound having the formula CH -NH in which R isL-()-d-amino-a-hydroxyvaleryl and R is OH or NH or a nontoxicpharmaceutically acceptable acid addition salt thereof; which processcomprises the consecutive steps of 5 A. acylating kanamycin A orkanamycin B with an acylating agent selected from the compounds havingthe formulas o 0 R! l 10 CH2 -O-C-O-N (or a carbodiimide thereof) or (ora carbodiimide thereof), in which R" and R are alike or different andeach is H, F, Cl, Br, N0 OH, (lower)alkyl or (lower)alkoxy, X is chloro,bromo or iodo, or a functional equivalent as an acylating agent; in aratio of one mole or less of acylating agent per 5 mole of kanamycin Aor B in a solvent, preferably selected from the group comprised ofdimethylformamide, dimethylacetamide, tetrahydrofuran, dioxane,1,2-dimethoxyethane, methanol, ethanol, water, acetone, pyridine,N-(lower)alkylpiperidine, or mixtures thereof, but preferablydimethylformamide-water, at a temperature below 50 C. and preferablybelow 25 C., to produce the compound having the formula CH -NH -Y inwhich Y is a radical of the formula prising OENQ' Go but preferably M isa radical selected from the group comprising -O-N l, mQNQ 4Q N02 2 -O-N@ -N l, but preferably ether, or the like but preferably water-ethyleneglycol dimethyl ether, to produce a compound of the formula in which R Yand W are as above; and

C. removing the blocking groups W and Y from compound by methodscommonly known in the art, and preferably, when W and Y are radicals ofthe formula by hydrogenating compound III with hydrogen in the presenceof a metal catalyst, preferably selected from the group comprisingpalladium, platinum, Raney nickel, rhodium, ruthenium and nickel, butpreferably palladium, and most preferably palladium on charcoal, in awater-water miscible solvent system, preferably selected from the groupcomprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethylether, propyleneglycol dimethyl ether, or the like, but preferably l:lwater-dioxane, and preferably in the presence of a catalytic amount ofglacial acetic acid to produce the compound of formula IV.

It should be apparent to those knowledgeable in the art that otheragents can be used in the process above to acylate the amine functionsof the intermediate compounds of the instant invention. This disclosureis meant to include all such acylating agents that produce labile amineblocking groups, said labile blocking groups commonly employed in thesynthesis of peptides. The labile blocking groups must be readilyremovable by methods commonly known in the art. Examples of said labileblocking groups and their removal can be found in the review of A.Kapoor, J. Pharm. Sciences, 59, pp. l-27 (1970). Functional equivalentsas acylating agent for primary amine groups would include correspondingcarboxylic chlorides, bromides, acid anhydrides, including mixedanhydrides and particularly the mixed anhydrides prepared from strongeracids such as the lower aliphatic monoesters of carbonic acid, of alkyland aryl sulfonic acids and of more hindered acids such asdiphenylacetic acid. In addition,

an acid azide or an active ester of thioester (e.g., withp-nitrophenol,2,4-dinitrophenol, thiophenol, thioacetic acid) may be used or the freeacid itself may be coupled with the kanamycin derivative (11) afterfirst reacting said free acid with N,N-di-methylchloroforminium chloride[cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI/6,360 1965)] or by the use of enzymes or of an N,N'-carbonyldiimidazole oran N,N'-carbonylditriazole [cf. Sheehan and Hess, J. Amer. Chem. Soc.,77, 1067, (1955)] or of alkynyla mine reagent [cf. R. Buijile and H. G.Viehe, Angew Chem., International Edition 3, 582 (1964)], or of aketenimine reagent [cf. C. L. Stevens and M. E. Monk, J. Amer. Chem.Soc, 83, 1010 (1961)]. Another equivalent of the acid chloride is acorresponding azolide, i.e., an amide of the corresponding acid whoseamide nitrogen is a member of a quasiaromatic five membered ringcontaining at least two nitrogen atoms, i.e., imidazole, pyrarole, thetriazoles, benzimidazole, benzotriazole and their substitutedderivatives. As an example of the general method for the preparation ofan azolide, N,N-carbonyldiimidazole is reacted with a carboxylic acid inequimolar proportions at room temperature in tetrahydrofuran chloroform,dimethylformamide, or a similar inert solvent to form the carboxylicacid imidazolide in practically quantitative yield with liberation ofcarbon dioxide and one mole of imidazole. Dicarboxylic acids yielddiimidazolides. The byproduct, imidazole, precipitates and may beseparated and the imidazolide isolated, but this is not essential. Thesereactions are well-known in the art (cf. U.S. Pat. Nos. 3,079,314,3,117,126 and 3,129,224 and British Pat. Nos. 932,644, 957,570 and959,054).

Compound IVa, 1-[L-(-)-8-amino-a-hydroxyvaleryl]-kanamycin A andcompound IVb, 1-[L-()- S-amino-a-hydroxyvaleryl]-I(anamycin B, possessexcellent anti-bacterial activity that appears superior in some respectsagainst certain pathogenic microorganisms than kanamycin A or Brespectively.

Illustrated below are two tables showing the minimal inhibitoryconcentration (MlCs) of kanamycin A to compound IVa (BB-K23) andkanamycin B compared to compound IVb (BB-K33) against a variety ofgrampositive and gram-negative bacteria as obtained by the Steersagar-dilution method. Mueller-Hinton agar medium was used in the studyof both tables.

TABLE I (MIC mgJml.)

BB-K23 Kanamycin MIC (meg/ml.) (lVa) A E. coli NII-IJ 1.6 0.8 JuhlA15119 1.6 1.6 A15l69 1.6 1.6 KM-R A20363 1.6 100 A9844 1.6 0.8 KM-RA20365 0.4 100 K-12 1.6 0.8 KM-R A20664 6.3 6.3 KM-R A20665 0.8 100 W677A20684 1.6 0.8 JR/W677 A20683 3.1 100 K. pneumoniae D-ll 0.8 0.2 Type 22No. 3038 A20680 3.1 100 S. marcescens A20019 3.1 1.6 P. aeruginosa DIS6.3 12.5 l-I9 D413 KM-R 25 100 A9923 6.3 50 A9930 0.8 12.5 A15150 12.5100 A15194 6.3 25 GM-R A20717 12.5 50 GM-R A20718 12.5 50 P. vulgarisA9436 0.4 0.4 A9526 0.8 0.4 P. mirabilis A9554 1.6 0.8 A9900 1.6 0.8 P.morganii A9553 0.8 0.8 A20031 0.8 0.8 S. aureus Smith 0.4 0.4 2091 SM-R3.1 1.6

TABLE I-Co:ntinued (MIC mg./ml.)

BB-K23 Kanamycin MIC (meg/ml.) (IVa) A KM-R A20239 3.1 100 Mycobacterium607 1.6 0.4 KMR 100 100 KM,SM-R 100 100 phlei 1.6 0.4 ranae 1.6 0.4

KM-R is kanamycin resistant. GM-R is gentamicin resistant. SM-R isstreptomycin resistant.

TABLE II (MIC mgjml.)

Kanamycin MIC (meg/ml.) BB-K33 BB-K34 B E. coli NIHJ 1.6 6.3 0.4

" Juhl A15169 1.6 6.3 0.8 KM-R H A20363 3.1 6.3 100 A9844 1.6 6.3 0.4KM-R I, H A20664 6.3 25 0.8

A20684 1.6 0.8 JR/W677 A20683 12.5 100 K. pneumoniae D-ll 0.4 1.6 0.1

" Type 22 3038 A20680 25 25 100 S. marcescens A20019 12.5 0.8 P.aeruginosa D-l5 3.1 12.5 6.3 H9 D-113 100 50 100 KM-R A15195 12.5 12.512.5 GM-R A2071? 12.5 GMR A20718 12.5 25 P. vulgaris 50 A9436 1.6 3.10.2

A9526 1.6 3.1 0.2 P. mirabilis A9900 1.6 6.3 0.8 P. morganii A20031 3.112.5 0.8 S. aureus Smith 0.4 1.6 0.1 209? SM-R 3.1 3.1 0.8 KM-R A202393.1 6.3 50

Mycobacterium 607 1.6 6.3 1.6

KM-R 100 100 KM,SM-R 100 100 100 phlei 0.4 6.3- 1.6 5 ranae 1.6 6.3 1.6

The above MIC data show that compounds Na and Nb are substantially moreactive against a number the test organisms, particularly the pseudomonasand other kanamycin resistant organisms.

The compounds IV are valuable as antibacterial agents, nutritionalsupplements in animal feeds, therapeutic agents in poultry and animals,including man, and are especially valuable in the treatment ofinfectious diseases caused by Gram-positive and Gram-negative bacteria.

The compound IV when administered orally are useful as an adjunctivetreatment for preoperative sterilization of the bowel. Both aerobic andanaerobic flora which are suseptible to these drugs are reduced in thelarge intestine. When accompanied by adequate mechanical cleansing, theyare useful in preparing for colonic surgery.

The compounds IV are effective in the treatment of systemic bacterialinfections in man when administered parenterally in the dosage range ofabout 250 mg. to about 3000 mg. per day in divided doses three or fourtimes a day. Generally the compounds are effective when administered ata dosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.

EXAMPLE 1 Preparation of L-8-Benzyloxycarbonylamino-a-hydroxyvalericacid (VI).

To a stirred solution of 400 mg (3.0 m moles) ofL-S-amino-a-hydroxyvaleric acid* and 250 mg (6.5 m moles) of sodiumhydroxide in 25 ml of water was added dropwise 580 mg (3.3 m moles) ofcarbobenzoxy chloride over a period of 30 minutes at 5C. The mixture wasstirred for an hour at l 5C, washed with 25 ml of ether, adjusted to pH2 with hydrochloric acid and extracted with three 30-ml portions ofether. The combined ethereal solution was shaken with ml of a saturatedsodium chloride solution, dried over anhydrous sodium sulfate andevaporated in vacuo to give crystals which were recrystallized frombenzene to yield 631 mg (78%) of VI, mp 110-111C.; infrared spectrum[lR(KBr)]: 3460, 3350, 1725, 1685, 1535, 1280, 730, 690cm. Nuclearmagnetic resonance spectrum [NMR(acetone-d 5 (in ppm) 1.70(4I-I, m)

4.14(2l-l, q, J=4.5l-Iz), 4.19(1H, m), 4.82(2I-l, s), 6.2(31-1, broad),7.25(5l-I, s). [01],, 1.6 (c 10, MeOl-l).

Anal Calcd for C H NO C, 58.42; I-I.6.41; N, 5.24.

Found: C, 58.36; H, 6.50; N, 5.27. *S. Ohshiro et al., Yakugaku Zasshi,87, 1184 (1967).

EXAMPLE 2 N-Hydroxysuccinimide ester ofL-y-benzyloxycarbonylamino-a-hydroxybutyric acid (VII) To a stirred andchilled solution of 535 mg (2.0 m moles) of VI and 230 mg (2.0 m moles)of N- hydroxysuccinimide in 55 ml of ethyl acetate was added 412 mg (2.0m moles) of N, N'-dicyclohexylcarbodiimide (DCC). The mixture wasstirred for 3 hours at room temperature and filtered to removeprecipitated N, N'-dicyclohexylurea. The filtrate was evaporated invacuo to yield 780 mg 100%) of viscous syrup (V11). 1R(Neat): 7 1810,1785, 1725 cm".

EXAMPLE 3 1-[L()-5-amino-a-hydroxyvaleryl] kanamycin A, [BB-K 23, Na]

To a stirred solution of 1.24 g (2.0 m moles) of kanamycin A (Ila) in 12ml of water. and,60 ml of 1,2-

dimethoxyethane (DME) was added dropwise 780 mg (2.0 m moles) of VII in10 ml of DME. The mixture was stirred overnight and then evaporated todryness in vacuo. The residue was treated with 20 ml of water and shakenwith two 30-m1 portions of water-saturated butanol. The combined butanollayer was evaporated in vacuo to give 1.36 g of solid, which wasdissolved in 10 ml of water, 10 ml of dioxane and 1 ml of acetic acid,and hydrogenated overnight with 200 mg of 10% palladium on charcoal atatmospheric pressure at ordinary temperature. The hydrogenated mixturewas filtered and the filtrate evaporated to dryness in vacuo. Theresidue was dissolved in 20 ml of water. The solution was passed througha column of Amberlite CG-50** (NH 15 ml), which was washed with ml ofwater and then eluted with 760 ml of 0.1N and 880 ml of 0.2 N NI-I O1-I.The eluate was collected in 10-ml fraction. Tube Nos. 109 to 172 whichupon thin layer chromatography (TLC)* showed Rf 0.18 (S-1 10, ninhydrin)were combined, evaporated in vacuo and freeze-dried to give 330 mg(28%)of IVa, mp 185 (dec). lR(KBr): 1635, 1570 cm.

Anal Calcd for C H N O H O: C, 43.57: H, 7.16; N, 10.59.

Found: C, 43.25; H, 7.12; N, 9.83. **Amberlite CG-SO is the tradenamefor the chromatographic grade of a weakly acidic cationic exchange resinof a carboxylicpolymethacrylic type. *TLC: silica gel plate, CHCl-MeOH-28% NH4OI-I-H2O (124:2:1).

EXAMPLE 4 (10:1) gave colorless prisms melting at 7879 C. l. G. W.Anderson et al., J. Am. Chem. Soc., 86, 1839 (1964).

EXAMPLE 5 Preparation of 6'-Carbobenzoxykanamycin A (Ila) A solution of42.5 g. m.moles) of kanamycin A free base in 450 ml. of water and 500ml. of dimethylformamide (DMF) was cooled below 0 C. and stirredvigorously. To the solution was added dropwise over a period of abouttwo hours a solution of 22.4 g. (90 m.mole) ofN-(benzyloxycarbonyloxy)succinimide in 500 ml. of DMF. The mixture wasstirred at 10 to 0 C. overnight and then at room temperature for oneday. The reaction mixture was evaporated under reduced pressure belowabout 50 C. The oily residue was dissolved in a mixture of 500 ml. waterand 500 m1. butanol, the mixture being filtered to remove insolublematerial and separated into two layers. The butanol and aqueous layerswere treated with butanol-saturated water (500 ml. X 2) andwater-saturated butanol (500 ml. X 2), respectively, using a techniquesimilar to counter current distribution. The three aqueous layers werecombined and evaporated to dryness under reduced pressure to give anoily residue, 21 part of which crystallized on standing at roomtemperature. To the residue including the crystals was added about ml.of methanol, which dissolved the oil and separated it from the crystals.After adding about 300 ml. of ethanol, the mixture was kept at roomtemperature overnight to give a crystalline mass which was collected byfiltration. 1t weighed 44 g. The product contained a small amount ofkanamycin A as indicated by thin layer chromatography usingn-propanolpyridine-acetic acidwater (15:10:3112) as the solvent systemand ninhydrin as the spray reagent.

The crude product was dissolved in 300 ml. of water and chromatographedon a column (30 mm. diameter) of CG-50 ion-exchange resin (NHf type, 500ml.). The column was irrigated with 0.1 N ammonium hydroxide solutionand the eluate was collected in l-ml. fraction. The desired product wascontained in tube numbers -100, while kanamycin A recovered fromslower-moving fractions and the position isomer(s) of the product seemedto be contained in the fastermoving fractions. The fractions 10-1 10were combined and evaporated to dryness under reduced pressure to give24.6 g. (45%) of a colorless product 6'-carbobenzoxykanamycin A (ll)[6-Cbz-kanamycin A], which began to melt and color at 204 C. anddecomposed at 212 C. with gas evolution. [a],, +106 (6 2, H O).

*Detected by anthrone-sulfuric acid.

The final product was found to be accompanied by two minor components byTLC with one of the solvent systems tested. However, the final productwas used without further purification for the preparation of BB-K23(1Va).

EXAMPLE 6 Preparation of 6-Carbobenzoxykanamycin B (11b) To a chilledsolution of 8.1 g. (0.0168 mole) of kanamycin B in 120 ml. of water and80 ml. of 1,2- dimethoxyethane was added dropwise with stirring asolution of 4.2 g. (0.0168 mole) of N- (benzyloxycarbonyloxy)succinimidein 40 ml. of 1,2- dimethoxyethane. The reaction mixture was stirredovernight and evaporated under reduced pressure. The residue wasdissolved in 100 ml. of water and shaken twice with 50 ml. ofwater-saturated n-butanol. The aqueous layer was separated and adsorbedon a column of 100 ml. of CG-50 (NH, type). The column was washed with200 ml. of water, eluted with 0.05 N NH OH. The eluate was collected inl0-ml. fraction. Fractions 121 to 180 were collected, evaporated andfreeze-dried to give 1.58 g. of the desired product. Fractions 1 to 120were evaporated and re chromatographed on CG-50 (NHJ) to give 1.21 g.(12%) of the product (11b). M.p. l5l-l52 C. (dec.). [a] +l04 (C. 2.5, H0). y ,l7l0 cmf.

Anal. calcd. for C H N O C, 50.56; H, 7.02; N,

Found: C, 50.71; H, 7.38; N, 11.48.

TLC (silica gel F254), RF 0.03 in n-PrOH-pyridine AcOH-H O (15:10:3112);Rf 0.16 in acetone-AcOH- H O (2016274).

EXAMPLE 7 Preparation of l[L()-6-A.mino-a-hydroxyvaleryl] kanamycinB,[BB-K 33 (lVb)] To a stirred solution of 618 mg (l m mole) of 11b in30 ml of water-DME (1:2) was added 364 mg (l m mole) of VII in 10 ml ofdry DME in one portion. The mixture was stirred overnight at roomtemperature and evaporated in vacuo. The aqueous concentrate wasextracted with two 20-m1 portions of water-saturated nbutanol. Thebutanol extracts were evaporated to dryness to give 530 mg of solid,which was dissolved in 40 ml of water-DME(1:1) and hydrogenatedovernight with mg of palladium on carbon at atmospheric pressure at roomtemperature. The catalyst was removed by filtration. The filtrate wasevaporated in vacuo to remove most of the organic solvent. The aqueoussolution was charged on a column of Amberlite CG-50(NH 14 ml), which waswashed with 140 ml of water and then eluted with 410 ml of 0.1 N, 760 mlof 0.2N, 650 ml of 0.5N and. 510 ml of 1.0 N NH OH. The eluate wascollected in lO-ml fraction. Tube Nos. 127 to 141 which showed Rf 0.22by TLC on silica gel plate (S-ll0, ninhydrin) were pooled, evaporated invacuo and lyophilized to give 103 mg (17%) of BB-K 33, mp 190C(dec).lR(KBr): y 1635 cm.

Anal Calcd for C H N O 2H CO C, 41.55; H, 6.97; C, 11.63.

Found: C, 41.44; H, 7.09; C, 11.75 Tube Nos. to 216 which showed Rf 0.11(S-110, ninhydrin) were combined, evaporated in vacuo and lyophilized togive 42 mg (7%) of BB-K 34, mp l80l85C (dec). IR (KBr): 7 1640 cm.

Anal Calcd for C2 H55N7O 4-4H2cO3: C, H, 6.60; N, 10.19.

Found: C, 39.57; H, 6.63; N, 10.64.

The identity of the compound BB-K34 was not established although it isknown that it is a diacylated derivative of kanamycin B possessing weakantibacterial activity as compared to kanamycin B and BB-K33 (lVb).

EXAMPLE 8 Preparation of the Monosulfate Salt ofl-[L-(-)-8-amino-ahydroxyvaleryllkanamycin A or One mole ofl-[L-()-8-amino-a-hydroxyvaleryl] kanamycin A or B is dissolved in 1 to3 liters of water. The solution is filtered to remove any undissolvedsolids. To the chilled and stirred solution is added one mole ofsulfuric acid dissolved in 500 ml. of water. The mixture is allowed tostir for 30 minutes, following which cold ethanol is added to themixture till precipitation occurs. The solids are collected byfiltration and are determined to be the desired monosulfate salt.

EXAMPLE 9 Preparation of the Disulfate Salt ofl-[L-()-6-amino-a-hydroxyvaleryllkanamycin A or Thirty-five grams of1-[L-()-8-amino-a-hydroxyvalcryllkanamycin A or B (as themonobicarbonate trihydrate) is dissolved in 125 ml. of deionized water.

The pH is adjusted to 7-75 with 50% V/\/ sulfuric acid. Eight and onehalf grams of Darco G-60 (activated charcoal) is added and the mixtureis slurried at ambient room temperature for 0.5 hour. The carbon isremoved by suitable filtration and washed with 40 ml. of water. Thewater wash is added to the filtrate.

The combined filtrate-wash above is adjusted to pH 2-2.6 with 50% V/Vsulfuric acid. A large amount of carbon dioxide evolves. The solution isleft at house vacuum with stirring for minutes to expel additionalcarbon dioxide.

Eight and one half grams of Darco G-60 is added to the degassedsolution. The mixture is slurried for 0.5 hour at ambient roomtemperature. The carbon is removed by suitable filtration and and washedwith 35 ml. of deionized water. The water is added to the filtrate.

The combined filtrate-wash is adjusted to pH ll.3 with 50% V/V sulfuricacid. This solution is added with rapid stirring over a 10 minute periodto 600800 ml. of methanol (3-4 volumes of methanol). The mixture isstirred for 5 minutes at pH ll.3, passed through a 100 mesh screen,stirred for 2 minutes and allowed to settle for 5 minutes. Most of thesupernatant is decanted. The remaining slurry is suitably filtered,washed with 200 ml. of methanol and vacuum dried at 50C. for 24 hours toyield the appropriate disulfate salt.

We claim:

1. A compound having the formula in which R is H, R isL-()-5-amino-a-hydroxyvaleryl and R is OH or NH or a nontoxicpharmaceutically acceptable salt thereof.

2. The compound of claim 1 wherein R is H, R is L-()-8-amino-a-hydroxyvaleryl and R is OH or a nontoxic pharmaceuticallyacceptable acid addition salt thereof.

3. The compound of claim 1 wherein R is H, R is L-()-8-amino-a-hydroxyvaleryl and R is Nl-l or a nontoxic pharmaceuticallyacceptalble acid addition salt thereof.-

4. The monosulfate salt of the compound of claim 2.

5. The monosulfate salt of the compound of claim 3.

6. The disulfate salt of the compound of claim 2.

7. The disulfate salt of the compound of claim 3.

1. A COMPOUND HAVING THE FORMULA
 2. The compound of claim 1 wherein R1is H, R2 is L-(-)- delta -amino- Alpha -hydroxyvaleryl and R3 is OH; ora nontoxic pharmaceutically acceptable acid addition salt thereof. 3.The compound of claim 1 wherein R1 is H, R2 is L-(-)- delta -amino-Alpha -hydroxyvaleryl and R3 is NH2; or a nontoxic pharmaceuticallyacceptalble acid addition salt thereof.
 4. The monosulfate salt of thecompound of claim
 2. 5. The monosulfate salt of the compound of claim 3.6. The disulfate salt of the compound of claim
 2. 7. The disulfate saltof the compound of claim 3.